Neutralizing antibodies are critical for vaccine-induced protection of many viral diseases. They act in most cases by blunting the initial infection, which is then resolved by cytotoxic T cells. In fact, several lines of evidence argue that both arms of the adaptive immune response are indeed required to resolve experimental retroviral infections (and most likely HIV infection). However, it has been extraordinarily difficult to elicit antibodies that neutralize primary field isolates of HIV-1. Thus, if one considers such antibodies to be an important defense against HIV-1 infection and disease, there remains a significant gap in the design of current HIV-1 vaccine candidates. Therefore, we have developed an alternative strategy to generate serum antibodies that neutralize primary isolates of HIV-1. This novel approach exploits the existence of several potent broadly neutralizing human monoclonal antibodies against HIV-1 and the unique gene-delivery properties of recombinant adeno-associated virus (rAAV) vectors, rAAV vectors have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes. Because of the flexibility of this system, light and heavy chain antibody genes can be incorporated into a single rAAV vector, and the antibody-expressing vector can then be used to transduce muscle in vivo. This, in turn, leads to sustained expression and secretion of biologically active antibody molecules from transduced myofibers. We have shown that the human monoclonal antibody IgG1b12 can be expressed in exactly this fashion, with significant levels of HIV neutralizing activity present in sera of mice for over 6 months after a single intramuscular administration of the vector. In Project 4, we will further refine and optimize this novel strategy by developing rAAV antibody vectors based on rAAV-1 and rAAV-5, with the hopes of increasing IgG production 10-100 fold. We will also investigate ways to enhance the specific activity of the antibody genes themselves by testing alternative antibody constructions (Fab or single-chain) that retain neutralizing activity, but may be more efficiently secreted than the full-length IgG molecule from skeletal myocytes. Lastly, the efficacy of this vaccine approach will be determined using a SHIV challenge model of HIV-1 infection.